Compositions and methods for therapy for diseases characterized by defective chloride transport

ABSTRACT

Compositions and methods for therapy of cystic fibrosis, asthma, and other conditions characterized by defective chloride transport are provided. The compositions comprise one or more compounds such as flavones and/or isoflavones, ascorbate and/or derivatives thereof capable of stimulating chloride transport in epithelial tissues. Therapeutic methods involve the administration (e.g., orally or via inhalation) of such compositions to a patient afflicted with cystic fibrosis, asthma, and/or another condition responsive to stimulation of chloride transport.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/769,619, filed Jan. 30, 2004, now U.S. Pat. No. 7,718,694, issued May18, 2010; which is a continuation-in-part of U.S. patent applicationSer. No. 09/982,315, filed Oct. 17, 2001, now U.S. Pat. No. 7,335,683,issued Feb. 26, 2008; which is a divisional of U.S. patent applicationSer. No. 09/174,077, filed Oct. 16, 1998, now U.S. Pat. No. 6,329,422,issued Dec. 11, 2001; which is a continuation-in-part of U.S. patentapplication Ser. No. 08/951,912, filed Oct. 16, 1997, now U.S. Pat. No.5,972,995, issued Oct. 26, 1999.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No. R01HL071829 awarded by the National Institutes of Health. The governmentmay have certain rights in this invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200116_(—)403C3_SEQUENCE_LISTING.txt. The textfile is 66 KB, was created on Mar. 11, 2010, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the treatment of diseasescharacterized by defective chloride transport, including cysticfibrosis, asthma, chronic obstructive pulmonary disease (COPD), andother inflammatory disorders of the airways, intestinal constipation,pancreatitis, and dry eye syndrome. The invention is more particularlyrelated to compositions comprising one or more compounds such asflavones and/or isoflavones, and/or vitamin C and related compounds,which may be used to activate chloride transport (i.e., absorptionand/or secretion) in epithelial tissues of the airways, the intestine,the pancreas and other exocrine glands.

2. Description of the Related Art

Cystic fibrosis is a lethal genetic disease afflicting approximately30,000 individuals in the United States. Approximately 1 in 2500Caucasians is born with the disease, making it the most common lethal,recessively inherited disease in that population.

Cystic fibrosis affects the secretory epithelia of a variety of tissues,altering the transport of water, salt and other solutes into and out ofthe blood stream. In particular, the ability of epithelial cells in theairways, liver, pancreas, small intestine, reproductive tract and othertissues to transport chloride ions, and accompanying sodium and water,is severely reduced in cystic fibrosis patients, resulting inrespiratory, pancreatic and intestinal ailments. The principle clinicalmanifestation of cystic fibrosis is the resulting respiratory disease,characterized by airway obstruction due to the presence of thick mucusthat is difficult to clear from airway surfaces. This thickened airwayliquid contributes to recurrent bacterial infections and progressivelyimpairs respiration, eventually resulting in death.

In cystic fibrosis, defective chloride transport is generally due to amutation in a chloride channel known as the cystic fibrosistransmembrane conductance regulator (CFTR; see Riordan et al., Science245:1066-73, 1989). CFTR is a linear chloride channel found in theplasma membrane of certain epithelial cells, where it regulates the flowof chloride ions in response to phosphorylation by a cyclicAMP-dependent kinase. Many mutations of CFTR have been reported, themost common of which is a deletion of phenylalanine at position 508(ΔF508-CFTR), which is present in approximately 70% of patients withcystic fibrosis. A glycine to aspartate substitution at position 551(G551D-CFTR) occurs in approximately 1% of cystic fibrosis patients.

Current treatments for cystic fibrosis generally focus on controllinginfection through antibiotic therapy and promoting mucus clearance byuse of postural drainage and chest percussion. However, even with suchtreatments, frequent hospitalization is often required as the diseaseprogresses. New therapies designed to increase chloride ion conductancein airway epithelial cells have been proposed, but their long termbeneficial effects have not been established and such therapies are notpresently available to patients.

Hypersecretion of sticky mucus by the airways is a hallmark ofinflammatory airway diseases, such as asthma, chronic bronchitis andCOPD (chronic obstructive airway disease). Asthma is currently aworldwide problem, with increasing prevalence in both children andadults. Total prevalence is estimated to be 7.2% of the world'spopulation (6% in adults, 10% in children). However, there can be widevariation between the prevalence of asthma in different countries andeven within different areas of a country. About 20 million Americansreport having asthma with more than 70% of people with asthma alsosuffering from allergies. Sixty percent of people with asthma sufferspecifically from allergic asthma. In 1999, it was estimated that 24.7million Americans have been diagnosed with asthma in their lifetime.Over six million children under 18 report having asthma.

Chronic bronchitis and COPD are commonly found in long-term smokerswhere excessive mucus secretions and poor mucociliary clearance causerecurring airway inflammation and destruction of the airway epithelium.Current treatment effects are limited and the prognosis of airwaydisease in long-term smokers is poor.

Accordingly, improvements are needed in the treatment of diseasescharacterized by defective chloride transport, such as cystic fibrosis,asthma, chronic obstructive pulmonary disease, and other inflammatorydisorders of the airways, intestinal constipation, pancreatitis, and dryeye syndrome. The present invention fulfills this need and furtherprovides other related advantages.

BRIEF SUMMARY OF THE INVENTION

Briefly stated, the present invention is directed to compositions andmethods for enhancing chloride transport in epithelial cells and for thetherapy of diseases characterized by defective cellular chloridetransport.

One aspect of the present invention is directed to a method for treatinga disease characterized by defective chloride transport in a mammalcomprising administering to the mammal one or more compounds selectedfrom the group consisting of ascorbic acid, ascorbate salts,dehydroascorbic acid and reservatrol. In one embodiment, the methodfurther includes administering one or more compounds including but notlimited to flavones and isoflavones, wherein the compound is capable ofstimulating chloride transport, and wherein the compound is notgenistein. In another embodiment, the compound includes one or more ofquercetin, apigenin, kaempferol, biochanin A, flavanone, flavone,dihydroxyflavone, trimethoxy-apigenin, apigenin 7-O-neohesperidoside,fisetin, rutin, daidzein and prunetin. In yet another embodiment, thedisease to be treated may be any one or more of asthma, cystic fibrosis,chronic obstructive pulmonary disease, intestinal constipation,pancreatitis, and dry eye syndrome. In certain embodiments, the compoundor compounds described herein are administered orally. In oneembodiment, the compounds of the present invention are administered byinhalation, or topically.

Another aspect of the present invention is directed to a method fortreating a disease characterized by defective chloride transport in amammal comprising; administering to the mammal one or more compoundsselected from the group consisting of ascorbic acid, ascorbate salts,dehydroascorbic acid and reservatrol; administering to the mammal one ormore compounds selected from the group consisting of flavones andisoflavones, wherein the compound is capable of stimulating chloridetransport; and administering to the mammal one or more compoundsselected from the group consisting of: a compound that increasesexpression of a CFTR in an epithelial cell; and a chemical chaperonethat increases trafficking of a CFTR to a plasma membrane in anepithelial cell. In one embodiment, the disease to be treated may becystic fibrosis or asthma. In a further embodiment, the compounds of thepresent invention to be administered are present within a compositioncomprising a physiologically acceptable carrier or excipient.

An additional aspect of the present invention is directed to a method oftreating diarrhea comprising, administering to a patient showingsymptoms of diarrhea, an effective amount of a compound that blocksvitamin C transport via SVCT1 and/or SVCT2 in intestinal epithelia. Inone embodiment, the method includes further administering to the patienta CFTR chloride channel blocker.

In another aspect of the present invention, a method for identifying anagent that alters chloride transport is provided, comprising; contactingan epithelial cell expressing SVCT1 and/or SVCT2 with a test agent andascorbate; measuring chloride transport in the epithelial cell contactedwith the test agent and ascorbate as compared to an epithelial cellcontacted with a control compound and ascorbate; wherein a statisticallysignificant increase or decrease in chloride transport in the cellcontacted with the test agent as compared to the chloride transport inthe cell contacted with control compound indicates the test agent alterschloride transport.

Another aspect of the present invention is directed to a compositioncomprising: (a) one or more flavones or isoflavones capable ofstimulating chloride secretion; (b) one or more of: (i) a compound thatincreases expression of a CFTR protein in an epithelial cell; and (ii) achemical chaperone that increases trafficking of a CFTR protein to aplasma membrane in an epithelial cell; (c) one or more of a compoundselected from the group consisting of ascorbic acid, ascorbate salts,dehydroascorbic acid and reservatrol; and (d) a physiologicallyacceptable carrier. In one embodiment, the compound of part (b)increases expression and/or trafficking of a mutated CFTR, such as aCFTR that has a mutation at position 551 and/or a CFTR that has a ΔF508mutation.

An additional aspect of the present invention is directed to a method ofidentifying an agent that stimulates chloride transport, comprising: (a)contacting, in the absence and presence of a candidate agent, (i) anascorbate compound, and (ii) a biological sample comprising a cell,under conditions and for a time sufficient to induce chloride transport;and (b) detecting chloride transport, wherein a level of detectablechloride transport that is increased in the presence of the candidateagent relative to the level of detectable chloride transport in theabsence of the agent indicates an agent that stimulates chloridetransport. In certain embodiments, the ascorbate compound is vitamin Cor a derivative thereof. In another embodiment the candidate agent is aflavonoid or an isoflavonoid. In an additional embodiment, the cell isan epithelial cell. In a further embodiment, the cell comprises at leastone transport molecule, such as a CFTR or a SVCT (e.g., SVCT1 or SVCT2).In one embodiment, the step of contacting does not increase a level ofintracellular cAMP.

Another aspect of the present invention provides a method of identifyingan agent that impairs chloride transport, comprising: (a) contacting, inthe absence and presence of a candidate agent, (i) an ascorbatecompound, and (ii) a biological sample comprising a cell, underconditions and for a time sufficient to induce chloride transport; and(b) detecting chloride transport, wherein a level of detectable chloridetransport that is decreased in the presence of the candidate agentrelative to the level of detectable chloride transport in the absence ofthe agent indicates an agent that impairs chloride transport. In oneembodiment, the ascorbate compound is vitamin C or a derivative thereof.In another embodiment, the candidate agent may be a flavonoid or anisoflavonoid. In a further embodiment, the cell is an epithelial cell.In yet another embodiment, the cell comprises at least one transportmolecule, such as a CFTR or a SVCT (e.g., SVCT1 or SVCT2). In oneembodiment, the step of contacting does not increase a level ofintracellular cAMP.

Within another aspect, the present invention provides methods forenhancing chloride transport in epithelial cells, comprising contactingepithelial cells with a compound selected from the group consisting offlavones and isoflavones, wherein the compound is capable of stimulatingchloride transport and wherein the compound is not genistein. Withincertain embodiments, the compound is (a) a polyphenolic compound havingthe general formula:

wherein carbon atoms at positions 2, 3, 5, 6, 7, 8, 2′, 3′, 4′, 5′ and6′ are bonded to a moiety independently selected from the groupconsisting of hydrogen atoms, hydroxyl groups and methoxyl groups, andwherein X is a single bond or a double bond; or (b) a stereoisomer orglycoside derivative of any of the foregoing polyphenolic compounds.Such compounds include, within certain embodiments, quercetin, apigenin,kaempferol, biochanin A, flavanone, flavone, dihydroxyflavone,trimethoxy-apigenin, apigenin 7-O-neohesperidoside, fisetin, rutin,daidzein and prunetin. For enhancing chloride transport in airwayepithelial cells of a mammal, compounds may be administered orally or byinhalation. Other epithelial cells that may be employed includeintestinal, pancreas, gallbladder, sweat duct, salivary gland andmammary epithelial cells. Within certain embodiments, the compound iscombined with a substance that increases expression of a CFTR; and/or achemical chaperone that increases trafficking of a CFTR to the plasmamembrane.

Within other aspects, methods for enhancing chloride transport inepithelial cells may comprise contacting epithelial cells with acompound selected from the group consisting of reservatrol, ascorbicacid, ascorbate salts and dehydroascorbic acid. Such compounds mayfurther be used in combination with a flavone or isoflavone as providedabove.

Within other aspects, the present invention provides methods fortreating cystic fibrosis in a patient, comprising administering to apatient a compound as described above, wherein the compound is capableof stimulating chloride transport. Within certain embodiments, thecompound is genistein, quercetin, apigenin, kaempferol, biochanin A,flavanone, flavone, dihydroxyflavone, trimethoxy-apigenin, apigenin7-O-neohesperidoside, fisetin, rutin, daidzein or prunetin. Within otherembodiments, the compound is reservatrol, ascorbic acid, ascorbate saltsand dehydroascorbic acid. Such compounds may be administered alone or incombination. Compounds may be administered orally or by inhalation.Within certain embodiments, the compound is combined with a substancethat increases expression of a CFTR; and/or a chemical chaperone thatincreases trafficking of a CFTR to the plasma membrane.

Within further related aspects, the present invention provides methodsfor increasing chloride ion conductance in airway epithelial cells of apatient afflicted with cystic fibrosis, wherein the patient's CFTRprotein has a deletion at position 508, the method comprisingadministering to a mammal one or more compounds as described above,wherein the compound is capable of stimulating chloride secretion in theairway epithelial cells.

Within still further related aspects, the present invention providesmethods for increasing chloride ion conductance in airway epithelialcells of a patient afflicted with cystic fibrosis, wherein the patient'sCFTR protein has a mutation at position 551, the method comprisingadministering to a mammal one or more compounds as described above,wherein the compound is capable of stimulating chloride secretion in theairway epithelial cells.

Within further aspects, pharmaceutical compositions for treatment ofcystic fibrosis are provided, comprising (a) one or more flavones orisoflavones capable of stimulating chloride transport and (b) one ormore of: (i) a compound that increases expression of a CFTR in anepithelial cell; and/or (ii) a chemical chaperone that increasestrafficking of a CFTR to a plasma membrane in an epithelial cell; and;and in combination with a pharmaceutically acceptable carrier. Withincertain embodiments, the flavone or isoflavone may be genistein,quercetin, apigenin, kaempferol, biochanin A, flavanone, flavone,dihydroxyflavone, trimethoxy-apigenin, apigenin 7-O-neohesperidoside,fisetin, rutin, daidzein and/or prunetin, in combination with apharmaceutically acceptable carrier.

Within still further aspects, a pharmaceutical composition for treatmentof cystic fibrosis may comprise: (a) a polyphenolic compound having thegeneral formula:

wherein carbon atoms at positions 2, 3, 5, 6, 7, 8, 2′, 3′, 4′, 5′ and6′ are bonded to a moiety independently selected from the groupconsisting of hydrogen atoms, hydroxyl groups and methoxyl groups, andwherein X is a single bond or a double bond; or a stereoisomer orglycoside derivative of any of the foregoing polyphenolic compounds; (b)a compound selected from the group consisting of reservatrol, ascorbicacid, ascorbate salts and dehydroascorbic acid; and (c) aphysiologically acceptable carrier.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a recording of transepithelial short-circuit current (Y axis)as a function of time (X axis), showing the effect of apigenin on thecurrent across a Calu-3 cell monolayer. Measurements were performed inan Ussing chamber, where the basolateral membrane was permeabilized withα-toxin and a chloride gradient was applied across the apical membraneas a driving force. Tissue was first stimulated with cAMP (100 μM).Apigenin (50 μM) was subsequently added as indicated. The horizontal barrepresents 100 seconds, and the vertical bar represents 12 μA/cm².

FIG. 2 is a recording showing the effect of quercetin on transepithelialshort-circuit current across a Calu-3 cell monolayer in an Ussingchamber, where the basolateral membrane was permeabilized with α-toxinand a chloride gradient was applied across the apical membrane as adriving force. Tissue was first stimulated with cAMP (100 μM). Quercetin(30 μM) was subsequently added as indicated. Bars are 140 seconds(horizontal) and 12 μA/cm² (vertical).

FIG. 3 is a recording illustrating the dose-dependent stimulation oftransepithelial chloride secretion by quercetin (in the amountsindicated) across a primary bovine tracheal epithelium. Amiloride (50μM) was added to block sodium transport as indicated. The CFTR channelblocker diphenylcarboxylate (DPC, 5 mM) was added as shown.

FIG. 4 is a recording showing the effect of biochanin A ontransepithelial short-circuit current across a Calu-3 cell monolayer inan Ussing chamber, where the basolateral membrane was permeabilized withα-toxin and a chloride gradient was applied across the apical membraneas a driving force. The tissue was first stimulated with forskolin (Fsk,10 μM). Subsequent addition of biochanin A (Bio, 100 and 300 μM) wassubsequently added as indicated.

FIG. 5 is a cell-attached single channel patch clamp recording from a3T3 cell expressing ΔF508-CFTR. The cell was treated with 10 μMforskolin as shown. Genistein (50 μM) and apigenin (50 μM), were addedwhere indicated by boxes. The holding potential was 75 mV, and channelopenings were upward.

FIG. 6 is a whole cell patch clamp recording on an airway epithelialcell homozygous for ΔF508-CFTR. Before the measurement, the cell wasincubated for 2 days in 5 mM 4-phenylbutyrate. 30 μM quercetin was addedwhere indicated by the box. Further stimulation by forskolin (10 μM) isalso shown. The holding potential was −60 mV.

FIG. 7 is a recording illustrating the effect of genistein on G551D-CFTRexpressed in a Xenopus oocyte. Current was measured with thetwo-electrode voltage clamp technique. G551D-CFTR was injected inoocyte. Current was first stimulated with forskolin (10 μM) andisobutylmethylxantine (IBMX; 2 mM). Genistein (50 μM) was added asindicated. The right panel shows current voltage relations recordedafter treatment with forskolin and IBMX (F/I) and after treatment withgenistein (F/I+Geni). A voltage ramp from −130 mV to +70 mV was appliedand current was recorded during the two conditions.

FIG. 8 is a recording illustrating the effect of quercetin on nasalpotential difference (PD) measurement in a healthy human volunteer.Amiloride (50 μM) was added to block sodium transport as indicated.Conditions were rendered chloride free (Cl free) and chloride secretionwas stimulated with isoproterenol (iso; 5 μM). Quercetin (querc; 10 μM)was added as indicated.

FIG. 9 is a recording illustrating the effect of apigenin and kaempferolon nasal PD in mice. Chloride secretion was stimulated withisoproterenol (iso; 5 μM), and amiloride (50 μM) was added to blocksodium transport as indicated. Under chloride-free conditions (Cl free),apigenin (50 μM, left panel) and kaempferol (kaemp, 50 μM, right panel)were added as indicated.

FIG. 10 is a recording illustrating the effect of genistein, with andwithout 4-phenylbutyrate, on chloride current in JME cells. Therecording was performed at 0 mV holding potential with a 17:150 mMchloride gradient from bath to pipette. The bottom trace is from anuntreated cell and the top trace is from a cell that had been incubatedin 5 mM 4-phenylbutyrate (4-PB) for two days. Forskolin (10 μM) andgenistein (30 μM) were added as indicated.

FIGS. 11A-11C are a whole cell patch clamp recording (FIG. 11A) andgraphs (FIGS. 11B and 11C) illustrating the effect of forskolin andgenistein on HeLa cells infected with a G551D-CFTR-containingadenovirus. Cells were stimulated with forskolin (10 μM) and genistein(30 μM), as indicated. The fit of the data with the Goldman equation isshown by the line in FIG. 11B. A current variance to mean current plotis shown in FIG. 11C.

FIGS. 12A and 12B illustrate the use of representative flavonoids forthe treatment of CF patients. FIG. 12A shows a recording from a patientwith the genotype G551D/ΔF508. Amiloride, chloride free solution andisoproterenol were added as indicated. The addition of genistein, asindicated, hyperpolarized nasal PD. FIG. 12B is a graph illustrating theaverage responses of nasal PD to genistein and quercetin of four CFpatients with the G551D mutation. The filled bars show, for comparison,the respective responses in healthy subjects.

FIGS. 13A-13C illustrate the effect of additional representativeflavonoids and isoflavonoids on chloride current in epithelial cells.FIG. 13A is a graph showing the stimulation of transepithelial chloridecurrents by reservatrol (100 μM), flavanone (100 μM), flavone (200 μM),apigenin (20 μM), apigenin 7-O-neohesperidoside (30 μM), kaempferol (20μM), fisetin (100 μM), quercetin (30 μM), rutin (30 μM), genistein (30μM), daidzein (50 μM), biochanin A (100 μM) and prunetin (100 μM) inCalu-3 monolayers. Experiments were performed in the presence of 10 μMforskolin. Stimulated currents are plotted relative to forskolinstimulated increase (forskolin stimulated currents are 100%). FIG. 13Bis a recording showing the effect of 7,4′-dihydroxyflavone on chloridecurrent in unstimulated tissue. This recording shows a dose-dependentstimulation of transepithelial short-circuit current (Isc) across Calu-3monolayers by 7,4′-dihydroxyflavone. Increasing concentrations of7,4′-dihydroxyflavone (as indicated in μM) were added to mucosal sideand dose-dependently stimulated chloride currents. Currents wererecorded with a serosal-to-mucosal chloride gradient at 0 mV and pulseswere obtained at 2 mV. FIG. 13C is a recording illustrating the effectof trimethoxy-apigenin. This recording shows dose-dependent stimulationof transepithelial short-circuit current (Isc) across Calu-3 monolayersby trimethoxy-apigenin. Increasing concentrations of trimethoxy-apigenin(as indicated in μM) were added to mucosal side and dose-dependentlystimulated chloride currents. Experiment was performed on unstimulatedtissue. Currents were recorded with a serosal-to-mucosal chloridegradient at 0 mV and pulses were obtained at 2 mV.

FIG. 14 is a recording illustrating the dose-dependent stimulation oftransepithelial short-circuit current (Isc) across Calu-3 monolayers byreservatrol. Increasing concentrations of reservatrol (as indicated inμM) were added to the mucosal perfusion and dose-dependently increasedchloride currents. For comparison, currents were further stimulated byserosal addition of 20 μM forskolin. Stimulated chloride current wascompletely blocked by addition of the chloride channel blocker DPC (5mM). Currents were recorded with a serosal-to-mucosal chloride gradientat 0 mV and pulses were obtained at 2 mV.

FIG. 15 is a recording showing L-ascorbic acid and genistein stimulationof transepithelial short-circuit current (Isc) across Calu-3 monolayers.Ascorbic acid (100 μM) was added as indicated. For comparison, ascorbicacid-stimulated chloride current was subsequently stimulated by the cAMPelevating agonist forskolin (20 μM, serosal). The CFTR activatorgenistein (20 mM) was then added to the mucosal perfusion as indicated.Stimulated current was completely blocked by addition of the chloridechannel blocker DPC (5 mM), added as indicated. Currents were recordedwith a serosal-to-mucosal chloride gradient at 0 mV and pulses wereobtained at 2 mV.

FIG. 16 is a recording showing L-Ascorbic acid and kaempferolstimulation of transepithelial short-circuit current (Isc) across Calu-3monolayers. 100 μM ascorbic acid and forskolin (fsk, 20 μM, serosal)were added as indicated. The CFTR activator kaempferol (20 μM) wassubsequently added, as indicated. Stimulated current was completelyblocked by addition of the chloride channel blocker DPC (5 mM). Currentswere recorded with a serosal-to-mucosal chloride gradient at 0 mV andpulses were obtained at 2 mV.

FIG. 17 is a recording illustrating the effect of L-ascorbic acid onnasal potential difference in human subjects. Amiloride, chloride-freesolution and L-ascorbic acid (100 μM) were added to the luminalperfusate in the nose. as indicated. The β-adrenergic agonistisoproterenol was also added as indicated. Stimulation was reversed bywashing out drugs with NaCl Ringer solution.

FIG. 18 is a recording illustrating the stimulation of transepithelialshort-circuit current (Isc) across Calu-3 monolayers by addition of 10,100 and 300 dehydroascorbic acid. Currents were recorded with aserosal-to-mucosal chloride gradient at 0 mV and pulses were obtained at2 mV.

FIG. 19 is a recording illustrating the stimulatory effect of 20genistein on transepithelial short-circuit current (Isc) across 31EG4mammary epithelial monolayers. Na currents were blocked by mucosaladdition of amiloride (10 mM), and chloride currents were furtherstimulated by forskolin (20 serosal), as indicated. Currents wererecorded in symmetrical NaCl Ringers solution at 0 mV and pulses wereobtained at 2 mV.

FIG. 20 shows the stimulation of CFTR activity by L-ascorbic acid. A.Activation of single CFTR Cl channels by L-ascorbic acid (100 bath) andforskolin (10 μM). Outside-out patch clamp recording from a Calu-3airway epithelial cell (150:15 mM Cl gradient from bath to pipette,holding potential=75 mV). B. P_(o) calculated from 20-s intervals fromrecording in A. C. Details of channel activity from recording in A. D.Single channel current-to-voltage relation, slope conductance is 8.9±0.2pS (n=4). E. Effects of increasing concentrations of L-ascorbic acid(∘), forskolin (●), and 100 nM forskolin in presence of 300 μML-ascorbate (300 μM Asc+Fsk,

) on intracellular cAMP levels.

FIG. 21 shows activation of Cl transport by L-ascorbic acid. A.Measurements of transepithelial Cl secretion across Calu-3 airwayepithelia in vitro. L-ascorbic acid (100 μM, mucosal) and forskolin (20μM, serosal) activated and DPC (4 mM) blocked Cl currents. B.Dose-dependency of ascorbate-stimulated Cl currents. Half-maximalstimulatory constant averaged 36.5±2.9 μM (n=14). C. Nasal potentialdifference (NPD) measurements in humans. L-ascorbic acid (300 μM) andthe cAMP agonist isoproterenol (10 μM) hyperpolarized NPD. Wash, washoutwith saline.

FIG. 22 shows Na-dependent vitamin C transporters in airway cells. A. Isa bar graph summary of ascorbate-stimulated Cl currents in Calu-3monolayers in the presence (+Na) or absence (−Na) of apical Na, or afterpre-treatment with 200 μM phloretin. * denotes significant differencefrom +Na, p≦0.05; n=6-14 experiments. B. RT-PCR analysis of SVCT1 andSVCT2 expression in hTE, Calu-3, and CF15 airway cells. GAPDH is shownas a control. 100-base pair size markers are indicated in lane 1.

FIG. 23 shows L-ascorbic acid is specific for CFTR-mediated Clsecretion. A. Transepithelial current (I_(SC)) across CF15 cells. Naabsorption was blocked by 20 μM amiloride. L-ascorbic acid (500 μM,mucosal) or forskolin (20 μM, serosal) stimulated Cl secretion inCFTR-corrected (+wtCFTR) but not in uncorrected CF epithelia (F508). B.Summary of ascorbate-stimulated Cl currents in the absence (Ascorbate)and presence of forskolin (Ascorbate+Forskolin). * significantlydifferent from ΔF508, p≦0.05; n=5-7 experiments.

FIG. 24 shows activation of ΔF508 CFTR by L-ascorbic acid andD-isoascorbic acid after correction of the trafficking defect in CFmice. A. Measurements of rectal potential difference (RPD) in micehomozygous for ΔF508 CFTR. Arrow indicates perfusion with L-ascorbate (1mM) or D-isoascorbate (300 μM). RPD was hyperpolarized by bothascorbates in CF mice treated with 4 mg/g TMAO for 24 hours (●, □), butnot in water-injected mice (∘). B. Summary of ascorbate-induced changesin RPD in TMAO-treated and control CF mice. ctrl, control; L-Asc, 1 mML-ascorbate; D-Iasc, 300 μM D-isoascorbate. * significantly differentfrom control CF mice, p≦0.05; n=3-9 experiments.

FIG. 25 shows activation of ΔF508 CFTR by L-ascorbic acid andD-isoascorbic acid after correction of the trafficking defect in CF15nasal epithelial cells using genetic or pharmacologic strategies. A-H.Measurements of transepithelial Cl currents (I_(SC)) inamiloride-treated CF15 monolayers homozygous for ΔF508 CFTR. Arrowindicates perfusion with L-ascorbate (1 mM) or D-isoascorbate (300 μM).I_(SC) was stimulated by both ascorbates in CF15 monolayersrecombinantly expressing wildtype CFTR (B,F) or were treated with 1 mMS-Nitrosoglutathione for 5-24 hours (C,G), but not in untreated CF nasalcells (A,E). D,H. Summary of ascorbate-induced changes in RPD inTMAO-treated and control CF mice. ctrl, control; L-Asc, 1 mML-ascorbate; D-Iasc, 300 μM D-isoascorbate. * significantly differentfrom untreated CF15 nasal epithelia, p≦0.05; n=2-13 experiments.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for the treatment of diseases characterized bydefective chloride transport, particularly with regard to defectivecellular chloride transport (e.g., defective export from or import tothe cell of chloride, such as chloride anion or in the form of achloride salt or other chloride-containing compound), for example, inepithelial tissues, including diseases such as cystic fibrosis, asthma,chronic obstructive pulmonary disease, and other inflammatory disordersof the airways, intestinal constipation, pancreatitis, and dry eyesyndrome and also including diseases that feature excessive accumulationof mucus, including cystic fibrosis, chronic bronchitis and asthma.Within the context of the present invention, defective chloridetransport may comprise a decrease (e.g., in a statistically significantmanner) or lack of chloride transport as compared to normalphysiological levels with which the skilled artisan will be familiar asa property of a particular cell or tissue type, organ system, or thelike, or an increase (e.g., in a statistically significant manner) inchloride transport as compared to such normal levels.

It has been found, within the context of the present invention, thatcertain flavones and isoflavones, as well as other polyphenoliccompounds, and ascorbate compounds such as ascorbic acid (vitamin C),and derivatives thereof, are capable of stimulating CFTR-mediatedchloride transport in epithelial tissues (e.g., tissues of the airways,intestine, pancreas and other exocrine glands) in a cyclic-AMPindependent manner. It has further been found, within the context of thepresent invention, that, under appropriate conditions, such compoundsmay stimulate chloride transport in certain cells, for example, cellshaving a mutated CFTR (e.g., ΔF508-CFTR or G551D-CFTR). Such therapeuticcompounds may be administered to patients suspected of having orafflicted with a disease characterized by defective chloride transport(e.g., defective cellular chloride transport) such as, cystic fibrosis,asthma, chronic obstructive pulmonary disease, and other inflammatorydisorders of the airways, intestinal constipation, pancreatitis, and dryeye syndrome as described herein.

The term “flavones,” as used herein refers to a compound based on thecore structure of flavone:

An “isoflavone” is an isomer of a flavone (i.e., the phenyl moiety atposition 2 is moved to position 3), and having the core structure shownbelow:

Certain flavones and isoflavones have the structure:

wherein carbon atoms at positions 2, 3, 5, 6, 7, 8, 2′, 3′, 4′, 5′ and6′ are bonded to a moiety independently selected from the groupconsisting of hydrogen atoms, hydroxyl groups and methoxyl groups, andwherein X is a single bond or a double bond. Stereoisomers and glycosidederivatives of such polyphenolic compounds may also be used within themethods provided herein.

Many flavones are naturally-occurring compounds, but synthetic flavonesand isoflavones are also encompassed by the present invention. A flavoneor isoflavone may be modified to comprise any of a variety of functionalgroups, such as hydroxyl and/or ether groups. Preferred flavonescomprise one or more hydroxyl groups, such as the trihydroxyflavoneapigenin, the tetrahydroxyflavone kaempferol and the pentahydroxyflavonequercetin. Preferred isoflavones comprise one or more hydroxyl and/ormethoxy groups, such as the methoxy, dihydroxy isoflavone biochanin A.Genistein is yet another preferred isoflavone for use within the methodsprovided herein.

Any flavone or isoflavone, or ascorbate compound (e.g., ascorbic acid,ascorbate, or a derivative thereof), that stimulates (e.g., increaseswith statistical significance) chloride transport within at least one ofthe assays described herein may be used for therapy of diseasescharacterized by defective chloride transport as provided hereinincluding, for example, cystic fibrosis and other diseases characterizedby abnormally high mucus accumulation in the airways, asthma, chronicobstructive pulmonary disease, and other inflammatory disorders of theairways, intestinal constipation, pancreatitis, and dry eye syndrome.Preferred therapeutic compounds include flavones and isoflavones thatoccur naturally in plants and are part of the human diet. Preferredcompounds include genistein (4′,5,7-trihydroxyisoflavone), as well asquercetin (3,3′,4′,5,7-pentahydroxyflavone), apigenin(4′5,7-trihydroxyflavone), kaempferol (3,4′,5,7-tetrahydroxyflavone) andbiochanin A (4′-methoxy-5,7-dihydroxyisoflavone), as depicted below:

Other suitable therapeutic compounds may be identified using therepresentative assays as described herein. Additional representativeflavones and isoflavones include flavanone, flavone, dihydroxyflavone,trimethoxy-apigenin, apigenin 7-O-neohesperidoside, fisetin, rutin,daidzein and prunetin. Representative flavones and isoflavones aresummarized in Tables I and II.

TABLE I Flavonoids No. Name X C3 C5 C7 C3’ C4’ 1 Apigenin ═ OH OH OH 2Apigenin7-O- ═ OH ONeo OH neohesperidoside 3 Dihydroxyflavone ═ OH OH 4Flavone ═ 5 Flavanone — 6 Fisetin ═ OH OH OH OH 7 Kaempferol ═ OH OH OHOH 8 Quercetin ═ OH OH OH OH OH 9 Rutin ═ ORut OH OH OH 10 Trimethoxy- ═H OCH3 OCH3 OCH3 apigenin where ═ a double bond, — is a single bond,ONeo is Neohesperidoside, ORut is rutinoside, OCH3 is methoxy, OH ishydroxy

TABLE II Isoflavonoids No. Name X C5 C7 C4’ 11 Biochanin ═ OH OH OCH3 12Daidzein ═ OH OH 13 Genistein ═ OH OH OH 14 Prunetin ═ OH OCH3 OH where═ a double bond, — is a single bond, ONeo is Neohesperidoside, ORut isrutinoside, OCH3 is methoxy, OH is hydroxy.

Genistein, quercetin, apigenin, kaempferol, biochanin A and otherflavones and isoflavones may generally be prepared using well knowntechniques, such as those described by Shakhova et al., Zh. Obshch.Khim. 32:390, 1962; Farooq et al., Arch. Pharm. 292:792, 1959; andIchikawa et al., Org. Prep. Prog. Int. 14:183, 1981. Alternatively, suchcompounds may be commercially available (e.g., from Indofine ChemicalCo., Inc., Somerville, N.J. or Sigma-Aldrich, St. Louis, Mo.). Furthermodifications to such compounds may be made using conventional organicchemistry techniques, which are well known to those of ordinary skill inthe art.

As noted above, other polyphenolic compounds may be used within themethods provided herein. For example, trihydroxystilbenes such asreservatrol (trans-3,5,4′-trihydroxystilbene) may be employed.Reservatrol is a polyphenolic compound having the following structure:

Other compounds that may be used within the methods provided herein areascorbate compounds such as ascorbic acid (vitamin C) and derivativesthereof. Accordingly and as provided herein, reference to ascorbate,ascorbic acid or vitamin C contemplates similar uses of other ascorbatederivatives having similar effects on chloride transport (e.g., cellularchloride transport) as those presently disclosed. Vitamin C, is arequired micronutrient for humans (Nishikimi, M., et al., (1994) J.Biol. Chem. 269, 13685-13688.) Dietary vitamin C acts as a cofactor forseveral intracellular enzymes and scavenges free radicals (Rumsey, S. C.et al., (1998) J. Nutr. Biochem. 9, 116-130.). In the lungs andrespiratory tract vitamin C mainly functions as an electron donor forreactive oxygen species (Willis, R. J., et al., (1976) Biochim. Biophys.Acta 444, 108-111; Slade, R., et al., (1993) Exp. Lung Res. 19, 469-484;van der Vliet, A., et al., (1999) Am. J. Physiol. 276, L289-L296; Kelly,F. J., et al., (1999) Lancet 354, 482-483). Epidemiological studiessuggested a link between high dietary intake of vitamin C and itsprotective effects against respiratory symptoms (Schwartz, J., et al.,(1994) Am. J. Clin. Nutr. 59, 110-114), however an alarming 25% of theU.S. population did not meet the recommended dietary intake levels forvitamin C as published in the Third National Health and NutritionExamination Survey (NHANES III, 1988-1994) (Food and Nutrition BoardInstitute of Medicine (2002) in Dietary Reference Intakes for Vitamin C,Vitamin E, Selenium, and Carotenoids (Natl. Acad. Press, Wash. DC), pp.95-185). Insufficient dietary intake of vitamin C (Kodavanti, et al.,(1996) Exp. Lung Res. 22(4), 435-448), environmental pollutants(Kodavanti, et al., Supra; Cross, et al., (1992) FEBS Lett. 298,269-272) and a number of inflammatory disorders of the airways are knownto severely deplete physiological pools of vitamin C in the respiratorytract. For example, low levels of vitamin C were found in patients withbronchial asthma (Kelly, et al., Supra; Menzel, D. B. (1992) Ann. N YAcad. Sci. 669, 141-155.), cystic fibrosis (Winklhofer-Roob, et al.,(1997) Am. J. Clin. Nutr. 65, 1858-66; Brown, et al., (1997) Am. J.Physiol. 273, L782-L788), chronic obstructive pulmonary disease(Calikoglu, et al., (2002) Clin. Chem. Lab. Med. 40, 1028-1031), acuterespiratory distress syndrome (Cross, et al., (1990) J. Lab. Clin. Med.115, 396-404), and smokers (Lykkesfeldt, et al., (2000) Am. J. Clin.Nutr. 71, 530-536) as well as in children exposed to environmentaltobacco smoke (Preston, et al., (2003) Am. J. Clin. Nutr. 77, 167-172).In addition to its well known function as an antioxidant in many speciesincluding mammalian species, in amphibians a regulatory function ofvitamin C on Cl transport across the cornea has been demonstrated(Scott, et al., (1975) Invest. Opthalmol. 14, 763-6).

The cystic fibrosis transmembrane conductance regulator chloride channel(CFTR) was cloned in 1989 (Riordan, et al., (1989) Science 245,1066-73). In the respiratory tract CFTR mediates the transport of Cl⁻across the epithelial cell apical membrane into the extracellular airwaysurface liquid (ASL), which transport is regulated to properly adjustASL salt composition. The dynamics of the ASL affect its physiologicalfunction, the most important of which is the removal of inhaledparticles and the support of mucociliary clearance (Widdicombe (1995)Am. J. Respir. Crit. Care Med. 151, 2088-2092). CF-like symptoms such asthickened airway secretions are often seen in chronic inflammatoryairway diseases without mutations in the CFTR gene, and there isemerging evidence that post-translational damage to CFTR by reactiveoxygen and nitrogen species decreases CFTR function (Bebok, et al.(2002) J. Biol. Chem. 277, 43041-43049).

As described in greater detail below and in the examples, theexperiments described herein were designed to investigate the role ofascorbate compounds such as vitamin C in controlling Cl⁻ secretion andto clarify the potential involvement of CFTR in the underlying Cl⁻conductance.

Accordingly, other compounds that may be used within the methodsprovided herein are ascorbic acid and derivatives thereof. Suchcompounds include L-ascorbic acid (L-xyloascorbic acid), dehydroascorbicacid (L-threo-2,3-Hexodiulosonic acid γ-lactone) and salts of theforegoing acids (e.g., L(+)-Ascorbic acid sodium salt, L(+)-Ascorbicacid iron(II) salt), as well as other ascorbate compounds as providedherein (e.g., L-Ascorbic acid 6-palmitate, L-Ascorbic acid 2-phosphatesesqui-magnesium salt, L-ascorbic acid 2-phosphate trisodium salt,L-Ascorbic acid 2-sulfate dipotassium salt) and other ascorbatecompounds known to the art (see for example, J Biol. Chem. 1999 Aug. 13;274(33): 23215-22; Yamamoto, et al., J Nutr Sci Vitaminol (Tokyo). 1992;Spec No: 161-4; Hornig D. Ann N Y Acad. Sci. 1975 Sep. 30; 258: 103-18).and which have detectable effects on chloride transport (e.g., cellularchloride transport) as herein disclosed.

Within certain preferred embodiments, ascorbic acid or a derivativethereof is used in combination with a polyphenolic compound as describedabove. Certain representative combinations include ascorbic acid and oneor more flavenoids and/or isoflavenoids (such as genistein and ascorbicacid; and kaempferol and ascorbic acid). Ascorbic acid may generally beused to treat or prevent genetic loss of chloride secretory function(e.g., cystic fibrosis), as well as other related loss or reducedchloride secretory function (e.g., intestinal constipation, dry eyesyndrome, asthma, and obstructive airway diseases).

Certain embodiments disclosed herein relate to the observation that theeffect of vitamin C on chloride transport depends on the function of thesodium-dependent vitamin C transporters (SVCT) (see in particularExample 13; the sequences for SVCTs are known in the art and areavailable, for example, via Genbank Accession Numbers SVCT1:NM_(—)005847 and SVCT2: AY380556). In certain embodiments, such as forthe treatment of diarrhea resulting from high dose vitamin C treatment,it may be desirable to block vitamin C transport in intestinalepithelial cells. As such, the present invention contemplates the use ofagents, including but not limited to, for example, phloretin andderivatives thereof, that block or otherwise decrease vitamin Ctransport in intestinal epithelia, in a statistically significantmanner, such as through SVCT1 and SVCT2. Accordingly, provided hereinare methods for identifying agents that alter chloride transport bycontacting a cell (e.g., an epithelial cell) such as a cell thatexpresses SVCT1 and/or SVCT2, with a test agent and an ascorbatecompound (e.g., vitamin C, ascorbic acid, ascorbate salts,dehydroascorbic acid or derivatives thereof) and measuring chloridetransport in the cell that has been contacted with the test agent andvitamin C as compared to chloride transport in a cell that has beencontacted with vitamin C in the absence of the test agent, for instance,in the presence of a control compound known not to affect chloridetransport. In this regard a statistically significant increase ordecrease in chloride transport in cells contacted with the test agentand vitamin C as compared to the cells contacted with vitamin C in theabsence of the test agent, or in the presence of a control compound,indicates the agent alters chloride transport. Such agents can beidentified by measuring chloride transport using any of the assaysdescribed herein and can also be identified using assays that screen forinhibition of SVCT1 and SVCT2 (e.g., by measuring vitamin C transport).Such agents can be identified using the assays as described herein, orknown in the art (Tsukaguchi, H., Tokui, T., Mackenzie, B., Berger, U.V., Chen, X. Z., Wang, Y., Brubaker, R. F. & Hediger, M. A. (1999)Nature 399, 70-75).

As used herein, epithelial cells include any cell of epithelioid originas known to those familiar with the art, and may be present in abiological sample as provided herein, for example a cell derived from amammalian organ outer layer (e.g., skin, renal cortical layer, etc.) oran organ lining layer (e.g., gastric epithelia, intestinal epithelia,etc.) such as mammalian epithelial cells that are capable of forming oneor more specialized structures, including desmosomes, tight junctions,adhesion plaques, distinct apical and basolateral regions, and the like.

Assays for Evaluating Chloride Transport

Flavones, isoflavones, ascorbate compounds such as ascorbic acid andderivatives thereof for use within the context of the present inventionhave the ability to stimulate chloride transport in epithelial tissues.Such transport may result in secretion or absorption of chloride ions.The ability to stimulate chloride transport may be assessed using any ofa variety of systems. For example, in vitro assays using a mammaliantrachea or a cell line, such as the permanent airway cell line Calu-3(ATCC Accession Number HTB55) may be employed. Alternatively, theability to stimulate chloride transport may be evaluated within an invivo assay employing a mammalian nasal epithelium. In general, theability to stimulate chloride transport may be assessed by evaluatingCFTR-mediated currents across a membrane by employing standard Ussingchamber (see Ussing and Zehrahn, Acta. Physiol. Scand. 23:110-127, 1951)or nasal potential difference measurements (see Knowles et al., Hum.Gene Therapy 6:445-455, 1995). Within such assays, a flavone orisoflavone that stimulates a statistically significant increase inchloride transport, in certain preferred embodiments, at a concentrationof about 1-10,000 μM, and in certain other preferred embodiments at aconcentration of 0.1-1,000 nM (and all concentrations therebetween) issaid to stimulate chloride transport. Generally, with regard to anascorbate compound such as ascorbic acid or a derivative thereof,stimulation of a statistically significant increase in chloridetransport, for example, at a concentration of about 1 to about 300 μMand in certain other preferred embodiments at a concentration of0.1-1,000 nM (and all concentration therebetween) is said to comprisestimulated chloride transport. In another embodiment, at least about 5%increase of chloride transport is considered beneficial. In certainembodiments, increased chloride transport by at least about 10%-15% maybe considered beneficial. In an additional embodiment, increasedchloride transport by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, is considered beneficial. Inother embodiments, it is considered beneficial if a compound, such asthose described herein, provides a stimulatory effect on chloridetransport at a level that is about 25%-60% of the level of chloridetransport that is stimulated using commonly known CFTR agonists (e.g.,forskolin). By way of non-limiting theory and as described herein, andin contrast to the situation that pertains to certain presently knownCFTR agonists such as forskolin, a known elevator of intracellular cAMP,according to certain embodiments the invention relates to chloridetransport that may be altered (e.g., increased or decreased in astatistically significant manner) under conditions that do not effect anincreased level of intracellular cAMP, which may refer to any detectableincreases in intracellular cAMP levels that are statisticallysignificant and that are typically greater than 5%, 10%, 20%, 30%, 40%,50% or more above the intracellular cAMP levels that exist in aparticular biological system prior to introduction of the conditionsthat alter chloride transport.

Certain embodiments of the invention as disclosed herein relate todetecting chloride transport, and in particular to detecting chloridetransport by cells, which may typically pertain to detection of cellularexport of intracellular chloride to the extracellular environment. Asdescribed herein and known in the art, CFTR represents an importanttransmembrane channel for chloride transport through the cellular plasmamembrane, and in humans CFTR is the major chloride transport channel.For example, certain single amino acid defects in CFTR manifestthemselves as lethal mutations in CF, underscoring the importance ofCFTR as a primary means for transporting chloride. Accordingly, basedupon the present disclosure any of a number of art acceptedmethodologies may be used for detecting chloride transport such asCFTR-mediated chloride transport. By way of illustration and notlimitation, descriptions of several such chloride transport assays,including in vitro and in vivo methodologies, are presented.

Within one in vitro assay, the level of chloride transport may beevaluated using mammalian pulmonary cell lines, such as Calu-3 cells, orprimary human, rat, mouse and bovine tracheal and alveolar cellcultures, as well as intestinal epithelial cell lines, such as T84,HT-29, Caco-2 and cell lines of other epithelial origin such as MDCK andFRT. In general, such assays employ cell monolayers, which may beprepared by standard cell culture techniques. Within such systems,CFTR-mediated chloride current may be monitored in an Ussing chamberusing intact epithelia. Anion efflux assays may be used to detectCFTR-mediated transport (e.g., iodide efflux, see for example RNA. 2003October; 9(10): 1290-7). Additionally, chloride transport (or generallyairway surface liquid composition), can be evaluated using any number ofindicators such as described in J Gen Physiol. 2003 November; 122(5):511-9. Alternatively, chloride transport may be evaluated usingepithelial tissue in which the basolateral membrane is permeabilizedwith anion-selective ionophores such as nystatin, amphotericin B orStaphylococcus aureus α-toxin, and in which a chloride gradient isimposed across the apical membrane (see Illek et al., Am. J. Physiol.270:C265-75, 1996). In either system, chloride transport is evaluated inthe presence and absence of a test compound (i.e., a flavone orisoflavone, or ascorbate or derivative thereof), and those compoundsthat stimulate chloride transport as described above may be used withinthe methods provided herein.

Within another in vitro assay for evaluating chloride transport, cellsare transfected with a chloride channel gene (e.g., CFTR) having amutation associated with cystic fibrosis. Any CFTR gene that is alteredrelative to the normal human sequence provided in SEQ ID NO:1, such thatthe encoded protein contains a mutation associated with cystic fibrosis,may be employed within such an assay. The most common disease-causingmutation in cystic fibrosis is a deletion of phenylalanine at position508 in the CFTR protein (ΔF508-CFTR; SEQ ID NO:4). Accordingly, the useof a CFTR gene encoding ΔF508-CFTR is preferred. However, genes encodingother altered CFTR proteins (e.g., G551D-CFTR; containing a glycine toaspartate point mutation at position 551; SEQ ID NO:6) may also be used.Cells such as NIH 3T3 fibroblasts may be transfected with an alteredCTFR gene, such as ΔF508-CFTR, using well known techniques (see Andersonet al., Science 25:679-682, 1991). The effect of a compound on chloridetransport in such cells may be evaluated by monitoring CFTR-mediatedcurrents using the patch clamp method (see Hamill et al., Pflugers Arch.391:85-100, 1981) with and without compound application.

Within another in vitro assay, a mutant CFTR may be microinjected intocells such as Xenopus oocytes. Chloride conductance mediated by the CFTRmutant in the presence and absence of a test compound (e.g., flavone,isoflavone, ascorbate or derivatives thereof) may be monitored with thetwo electrode voltage clamp method (see Miledi et al., Proc. R. Soc.Lond. Biol. 218:481-484, 1983).

Alternatively, such assays may be performed using a mammalian trachea,such as a primary human, mouse, sheep, pig or cow tracheal epitheliumusing the Ussing chamber technique as described above. Such assays areperformed in the presence and absence of test compound to identifyflavones, isoflavones, ascorbate or derivatives thereof that stimulatechloride transport.

Any of the assays described herein may be performed in the presence ofcAMP agonists known in the art and as described herein (see Example 8and other examples herein).

Any of the above assays may be performed following pretreatment of thecells with a substance that increases the concentration of CFTR mutantsin the plasma membrane. Such substances include chemical chaperones,which support correct trafficking of the mutant CFTR to the membrane,and compounds that increase expression of CFTR in the cell (e.g.,transcriptional activators). A “chemical chaperone,” as used herein isany molecule that increases trafficking of proteins to a cell membrane.More specifically, a chemical chaperone within the context of thepresent invention increases trafficking of a mutant CFTR (e.g., theΔ508-CFTR and/or G551D-CFTR) to the membrane by a statisticallysignificant amount. Chemical chaperones for use herein include, but arenot limited to, glycerol, dimethylsulfoxide, trimethylamine N-oxide,taurin, methylamine and deoxyspergualin (see Brown et al., Cell StressChaperones 1:117-125, 1996; Jiang et al., Amer J. Physiol.-Cell Physiol.44:C171-C178, 1998). Compounds that increase expression of CFTR in thecell include 4-phenylbutyrate (Rubenstein et al., J. Clin. Invest.100:2457-2465, 1997), sodium butyrate (Cheng et al., Am. J. Physiol.268:L615-624, 1995) and S-Nitrosoglutathione (Zaman, et al., BiochemBiophys Res Commun 284: 65-70, 2001; Snyder, et al., American Journal ofRespiratory and Critical Care Medicine 165: 922-6, 2002; Andersson, etal. Biochemical and Biophysical Research Communication 297(3): 552-557,2002.). Other compounds that increase the level of CFTR in the plasmamembrane (by increasing correct trafficking and/or expression of theCFTR) may be readily identified using well known techniques, such asWestern Blotting and immunohistochemical techniques, to monitormaturation of CFTR and evaluate effects on levels of plasma membraneCFTR, respectively.

In vivo, chloride secretion may be assessed using measurements of nasalpotential differences in a mammal, such as a human or a mouse. Suchmeasurements may be performed on the inferior surface of the inferiorturbinate following treatment of the mucosal surface with a testcompound during perfusion with the sodium transport blocker amiloride inchloride-free solution. The nasal potential difference is measured asthe electrical potential measured on the nasal mucosa with respect to askin electrode placed on a slightly scratched skin part (see Alton etal., Eur. Respir. J. 3:922-926, 1990) or with respect to a subcutaneousneedle (see Knowles et al., Hum. Gene Therapy 6:445-455, 1995). Nasalpotential difference is evaluated in the presence and absence of testcompound, and those compounds that results in a statisticallysignificant increase in nasal potential difference stimulate chloridetransport.

Compounds as provided herein may generally be used to increase chloridetransport within any of a variety of CFTR-expressing epithelial cells.CFTR is expressed in may epithelial cells, including intestinal, airway,lung, pancreas, gallbladder, kidney, sweat gland, lacrimal gland,salivary gland, mammary epithelia, epithelia of the male and femalereproductive tract, and non epithelial cells including lymphocytes. Allsuch CFTR-expressing organs are subject to stimulation by the compoundsprovided herein.

Accordingly, certain embodiments of the invention relate to a method ofidentifying an agent that stimulates (or impairs, and in either event ina statistically significant manner) chloride transport (e.g., cellulartransport of chloride across a plasma membrane), comprising contactingin the absence and presence of a candidate agent an ascorbate compoundand a biological sample comprising a cell, under conditions and for atime sufficient to induce chloride transport; and detecting chloridetransport, wherein a level of detectable chloride transport that isincreased in the presence of the candidate agent relative to the levelof detectable chloride transport in the absence of the agent indicatesan agent that stimulates chloride transport (and wherein, similarly, alevel of detectable chloride transport that is decreased in the presenceof the candidate agent relative to the level of detectable chloridetransport in the absence of the agent indicates an agent that impairschloride transport). In certain related further embodiments theascorbate compound may be vitamin C or a derivative thereof, and incertain other related further embodiments the candidate agent may be aflavonoid. In certain other related embodiments the cell comprises aCFTR and/or a vitamin C TR. In certain preferred embodiments the step ofcontacting does not increase the intracellular cAMP level.

Certain chloride transport assays may be adapted to a high throughputscreening (HTS) format and thus may be especially suited to automatedscreening of large numbers of candidate agents for activity in assays ofchloride transport. HTS has particular value, for example, in screeningsynthetic or natural product libraries for active compounds. The methodsof the present invention are therefore amenable to automated,cost-effective high throughput drug screening and have immediateapplication in a broad range of pharmaceutical drug developmentprograms. In a preferred embodiment of the invention, the compounds tobe screened are organized in a high throughput screening format such asa 96-well plate format, or other regular two dimensional array, such asa 384-well, 48-well or 24-well plate format or an array of test tubes.For high throughput screening the format is therefore preferablyamenable to automation. It is preferred, for example, that an automatedapparatus for use according to high throughput screening embodiments ofthe present invention is under the control of a computer or otherprogrammable controller. The controller can continuously monitor theresults of each step of the process, and can automatically alter thetesting paradigm in response to those results.

Typically, and in preferred embodiments such as for high throughputscreening, candidate agents are provided as “libraries” or collectionsof compounds, compositions or molecules. Such molecules typicallyinclude compounds known in the art as “small molecules” and havingmolecular weights less than 10⁵ daltons, preferably less than 10⁴daltons and still more preferably less than 10³ daltons. For example,members of a library of test compounds can be administered to aplurality of samples in each of a plurality of reaction vessels in ahigh throughput screening array as provided herein, each containing atleast one cell and being present in a form that permits conditions forinducing detectable chloride transport according to principles describedherein. Candidate agents further may be provided as members of acombinatorial library, which preferably includes synthetic agentsprepared according to a plurality of predetermined chemical reactionsperformed in a plurality of reaction vessels. For example, variousstarting compounds may be prepared employing one or more of solid-phasesynthesis, recorded random mix methodologies and recorded reaction splittechniques that permit a given constituent to traceably undergo aplurality of permutations and/or combinations of reaction conditions.The resulting products comprise a library that can be screened followedby iterative selection and synthesis procedures, such as a syntheticcombinatorial library of peptides (see e.g., PCT/US91/08694 andPCT/US91/04666) or other compositions that may include small moleculesas provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No.5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629). Thosehaving ordinary skill in the art will appreciate that a diverseassortment of such libraries may be prepared according to establishedprocedures, and tested for effect on chloride transport according to thepresent disclosure, using a biological sample (e.g., a cell such as anepithelial cell, or other suitable preparation of a cell, tissue, organ,or the like, for instance primary cell cultures, biopsy cells, tissueexplants, established cell lines including transformed, immortal orimmortalized cells, or other naturally occurring or geneticallyengineered cells or artificial cells).

Compositions and Methods of Use

For in vivo use, a compound as described herein is generallyincorporated into a pharmaceutical composition prior to administration.Within such compositions, one or more therapeutic compounds as describedherein are present as active ingredient(s) (i.e., are present at levelssufficient to provide a statistically significant effect on nasalpotential difference, as measured using a representative assay asprovided herein). A pharmaceutical composition comprises one or moresuch compounds in combination with any physiologically acceptablecarrier(s) and/or excipient(s) known to those skilled in the art to besuitable for the particular mode of administration. In addition, otherpharmaceutically active ingredients (including other therapeutic agents)may, but need not, be present within the composition.

In certain embodiments, an ascorbate compound or a derivative thereofand a flavonoid or isoflavonoid can be combined to additively open CFTRand thereby stimulate chloride transport. Within certain methodsprovided herein, a flavone, isoflavone, or derivative thereof, ascorbicacid or derivatives thereof, alone or in combination, may be combinedwith a substance that increases the concentration of CFTR mutants in theplasma membrane of a cell. As noted above, such substances may include achemical chaperone, which supports correct trafficking of the mutantCFTR to the membrane, and may also include compounds that increaseexpression of CFTR in the membrane. These substances may be containedwithin the same pharmaceutical composition or may be administeredseparately. Preferred chemical chaperones include, for example,glycerol, dimethylsulfoxide, trimethylamine N-oxide, taurin, methylamineand deoxyspergualin, or the like, and compounds that increase expressionof CFTR in the membrane include 4-phenylbutyrate and sodium butyrate, orthe like. The use of flavonoid and/or isoflavonoid compounds, orascorbic acid and derivatives thereof, as described herein, incombination with such substances may increase mutant CFTR activity, andameliorate symptoms of diseases associated with defective chloridetransport, such as cystic fibrosis.

In certain embodiments, the compounds described herein may be used totreat diarrhea, in particular diarrhea associated with vitamin Ctreatment. By way of non-limiting theory, CFTR blockers have beenemployed in the treatment of diarrhea (e.g., U.S. Pat. No. 5,234,922)whereas according to the instant disclosure and as described herein,agents that decrease vitamin C transporter activity are for the firsttime recognized as effecting decreased CFTR-mediated chloride transport.As such, the compositions, such as compositions comprising compoundsthat block vitamin C transporters (e.g., SVCT1 and/or SVCT2), can beadministered alone or in combination with CFTR blockers. Suchcompositions as well as other compositions described herein may be incombination with any physiologically acceptable carrier(s) and/orexcipient(s) known to those skilled in the art to be suitable for theparticular mode of administration.

Administration may be achieved by a variety of different routes.Preferred are methods in which the therapeutic compound(s) are directlyadministered as a pressurized aerosol or nebulized formulation to thepatient's lungs via inhalation. Such formulations may contain any of avariety of known aerosol propellants useful for endopulmonary and/orintranasal inhalation administration. In addition, water may be present,with or without any of a variety of cosolvents, surfactants, stabilizers(e.g., antioxidants, chelating agents, inert gases and buffers). Forcompositions to be administered from multiple dose containers,antimicrobial agents are typically added. Such compositions are alsogenerally filtered and sterilized, and may be lyophilized to provideenhanced stability and to improve solubility.

Another preferred route is oral administration of a composition such asa pill, capsule or suspension. Such compositions may be preparedaccording to any method known in the art, and may comprise any of avariety of inactive ingredients. Suitable excipients for use within suchcompositions include inert diluents (which may be solid materials,aqueous solutions and/or oils) such as calcium or sodium carbonate,lactose, calcium or sodium phosphate, water, arachis oil, peanut oilliquid paraffin or olive oil; granulating and disintegrating agents suchas maize starch, gelatin or acacia and/or lubricating agents such asmagnesium stearate, stearic acid or talc. Other inactive ingredientsthat may, but need not, be present include one or more suspending agents(e.g., sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia), thickeners (e.g., beeswax, paraffin or cetylalcohol), dispersing or wetting agents, preservatives (e.g.,antioxidants such as ascorbic acid), coloring agents, sweetening agentsand/or flavoring agents.

A pharmaceutical composition may be prepared with carriers that protectactive ingredients against rapid elimination from the body, such as timerelease formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems, and biodegradable, biocompatible polymers, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid,polyorthoesters, polylactic acid and others known to those of ordinaryskill in the art.

In general, the compositions of the present invention may beadministered by the topical, transdermal, oral, rectal (e.g., viasuppository or enema) or parenteral (e.g., intravenous, subcutaneous orintramuscular) route. In addition, the compositions may be incorporatedinto biodegradable polymers allowing for sustained release of thecomposition, the polymers being implanted in the vicinity of wheredelivery is desired, for example, in the intestinal epithelia. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al. J. Neurosurg. 74:441-446 (1991).

Pharmaceutical compositions are administered in an amount, and with afrequency, that is effective to inhibit or alleviate the symptoms of adisease characterized by defective chloride transport, such as cysticfibrosis, asthma, chronic obstructive pulmonary disease, and otherinflammatory disorders of the airways, intestinal constipation,pancreatitis, and dry eye syndrome, and/or to delay the progression ofthe disease. The effect of a treatment may be clinically determined bynasal potential difference measurements, or other measurements asdescribed herein and known in the art. The precise dosage and durationof treatment may be determined empirically using known testing protocolsor by testing the compositions in model systems known in the art andextrapolating therefrom. Dosages may also vary with the severity of thedisease. A pharmaceutical composition is generally formulated andadministered to exert a therapeutically useful effect while minimizingundesirable side effects. In general, an oral dose ranges from about 200mg to about 2000 mg, which may be administered 1 to 3 times per day.Compositions administered as an aerosol are generally designed toprovide a final concentration of about 10 to 50 μM at the airwaysurface, and may be administered 1 to 3 times per day. It will beapparent that, for any particular subject, specific dosage regimens maybe adjusted over time according to the individual need.

As noted above, a pharmaceutical composition may be administered to amammal to stimulate chloride transport, and to treat diseasescharacterized by defective chloride transport, such as cystic fibrosis,asthma, chronic obstructive pulmonary disease, and other inflammatorydisorders of the airways, intestinal constipation, pancreatitis, and dryeye syndrome and diseases with excessive accumulation of mucus,including cystic fibrosis, chronic bronchitis and asthma and to preventlocal vitamin C deficits in the respiratory tract due to oxidatativestress (ozone, tobacco smoke, air pollution, allergic reactions, and thelike). Patients that may benefit from administration of a therapeuticcompound as described herein are those afflicted with one or morediseases characterized by defective chloride transport, such as cysticfibrosis, asthma, chronic obstructive pulmonary disease, and otherinflammatory disorders of the airways, intestinal constipation,pancreatitis, and dry eye syndrome and diseases with excessiveaccumulation of mucus, including cystic fibrosis, chronic bronchitis andasthma. Such patients may be identified based on standard criteria thatare well known in the art, including the presence of abnormally highsalt concentrations in the sweat test, the presence of high nasalpotentials, or the presence of a cystic fibrosis-associated mutation.

Summary of Sequence Listing

SEQ ID NO:1 is a DNA sequence encoding human CFTR.

SEQ ID NO:2 is an amino acid sequence of human CFTR.

SEQ ID NO:3 is a DNA sequence encoding human CFTR with the ΔF508mutation.

SEQ ID NO:4 is an amino acid sequence of human CFTR with the ΔF508mutation.

SEQ ID NO:5 is a DNA sequence encoding human CFTR with the G551Dmutation.

SEQ ID NO:6 is an amino acid sequence of human CFTR with the G551Dmutation.

SEQ ID NOs:7-10 are PCR primers.

SEQ ID NO:11 is the polynucleotide of SVCT2 from human trachea epitheliaas set forth in GenBank accession No. AY380556 and is identical to thesequence set forth in SEQ ID NO:12 with the exception of one nucleotideexchange at position 1807 T->C.

SEQ ID NO:12) is the polynucleotide of SVC2 from human kidney as setforth in GenBank accession No. AJ269478.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Stimulation of Chloride Transport by RepresentativeFlavones and Isoflavones in Airway Cells

This Example illustrates the use of the representative compoundsapigenin, quercetin and biochanin A to enhance chloride secretion inCalu-3 human pulmonary cultures or in primary bovine tracheal cultures.

A Calu-3 cell monolayer was prepared in an Ussing chamber as describedby Illek et al., Am. J. Physiol. 270:C265-275, 1996. The basolateralmembrane was permeabilized with α-toxin and a chloride gradient wasapplied across the apical membrane as a driving force (see Illek et al,Am. J. Physiol. 270:C265-C275, 1996). The tissue was first stimulatedwith cAMP (100 μM), and then with a representative flavone orisoflavone.

As shown in FIGS. 1 and 2, subsequent addition of apigenin or quercetinfurther stimulated chloride current. FIG. 1 illustrates the shortcircuit current across the Calu-3 cell monolayer before and afteraddition of apigenin (50 μM). FIG. 2 illustrates the effect of quercetin(30 μM) on chloride current across a Calu-3 monolayer. In both cases,the flavone stimulated chloride current beyond the stimulation achievedby cAMP.

FIG. 3 illustrates the results of a related experiment to evaluate thedose-dependent stimulation of transepithelial chloride secretion byquercetin across a primary bovine tracheal epithelium. The epithelialcells were first treated with amiloride (50 μM), and then with quercetinat the indicated concentrations. The dose-response relation yielded ahalf maximal stimulation at 12.5 μM. At high concentrations ofquercetin, the current was blocked. Current was fully inhibited by theCFTR channel blocker diphenylcarboxylate (DPC, 5 mM).

To evaluate the effect of biochanin A, a Calu-3 cell monolayer wasprepared and permeabilized as described above. The tissue was firststimulated with forskolin (Fsk, 10 μM). The effect of biochanin A (Bio,100 and 300 μM) on short-circuit current (I_(SC)) across the Calu-3monolayer was evaluated in an Ussing chamber. As shown in FIG. 4,biochanin A further stimulated chloride secretion.

Example 2 Activation of Mutant CFTR by Representative Flavones andIsoflavones

This Example illustrates the use of the representative compoundsapigenin, quercetin and genistein to activate ΔF508-CFTR and G551D-CFTRin different cell types.

A cell-attached single channel patch clamp recording was obtained from a3T3 cell expressing ΔF508-CFTR as described by Hamill et al., PflugersArch. 391:85-100, 1981 and Fischer and Machen, J. Gen. Physiol.104:541-566, 1994. As shown in FIG. 5, stimulation of the cell with 10μM forskolin did not activate ΔF508-CFTR channel, but addition ofgenistein (50 μM) or apigenin (50 μM, where indicated by boxes) inducedΔF508-CFTR channel openings, and removal of these compounds inactivatedthe channels. The holding potential was 75 mV, and channel openings wereupward.

FIG. 6 presents a whole cell patch clamp recording on an airwayepithelial cell homozygous for ΔF508-CFTR (cell type: JME cell, seeJeffersen et al., Am. J. Physiol. 259:L496-L505, 1990). Before themeasurement, the cell was incubated for 2 days in 5 mM 4-phenylbutyrateto enhance ΔF508-CFTR expression in the plasma membrane (Rubenstein &Zeitlin, Ped. Pulm. Suppl. 12:234, 1995). Measurements were performed asdescribed by Fischer et al., J. Physiol. Lond. 489:745-754, 1995.Addition of 30 μM quercetin activated chloride current in the whole cellmode, which was further stimulated by forskolin. The holding potentialwas −60 mV.

The effect of genistein on chloride current in a Xenopus oocyteexpressing G551D-CFTR was measured with the two-electrode voltage clamptechnique (see Miledi et al., Proc. R. Soc. Lond. Biol. 218:481-484,1983). G551D-CFTR (2 ng in 50 mL of water) was injected into the oocyte.Current was first stimulated with forskolin (10 μM) andisobutylmethylxantine (IBMX; 2 mM). Genistein (50 μM) was found tofurther activate chloride currents. As shown in FIG. 7, genisteinincreased conductance and shifted reversal potential to the right, whichis indicative of a stimulated chloride current.

Example 3 Effect of Representative Flavones on Nasal PotentialDifference

This Example illustrates the in vivo use of quercetin, apigenin andkaempferol to activate the nasal potential difference in humans andmice.

The effect of quercetin on nasal potential difference (PD) measurementin a healthy human volunteer was measured as described by Knowles etal., Hum. Gene Therapy 6:445-455, 1995. Under conditions where sodiumtransport was blocked with amiloride (50 μM) and chloride secretion wasstimulated under chloride-free conditions with isoproterenol (5 μM),quercetin (10 μM) stimulated nasal PD further (FIG. 8).

The effect of apigenin and kaempferol on nasal PD in mice was evaluatedusing a method similar to that employed for measurements in humans,except that a plastic tube of approximately 0.1 mm diameter was used asan exploring nasal electrode. The plastic tube was perfused with testsolutions at approximately 10 μL/min. After blocking sodium transportwith amiloride (50 μM) and during stimulation of chloride secretion withisoproterenol (iso; 5 μM) under chloride-free conditions, apigenin (50μM, left panel) and kaempferol (kaemp, 50 μM, right panel) furtherstimulated nasal PD.

These results show that the representative flavenoids quercetin,apigenin, kaempferol and biochanin A stimulate chloride transport acrossepithelial tissues derived from the airways in vitro, and across nasalepithelium in vivo. The results also show that the CFTR mutants ΔF508and G551D can be activated by the representative compounds genistein andapigenin.

Example 4 Effect of Genistein on Chloride Current in Cells Expressing aMutant CFTR

This Example illustrates the ability of the representative isoflavonegenistein to activate chloride current in cells expressing a mutantCFTR.

In one experiment, genistein was used in combination with4-phenylbutyrate. Chloride current was measured in JME cells (humannasal epithelial cell line homozygous for the Δ508 mutation of CFTR; seeJefferson et al., Am. J. Physiol. 259:L496-505, 1990). The recording wasperformed at 0 mV holding potential with a 17:150 mM chloride gradientfrom bath to pipette. Under these conditions, the recorded current,shown in FIG. 10, is chloride current (Illek and Fischer, Am. J.Physiol. (Lung Cell. Mol. Physiol.):L902-910, 1998). The bottom trace inFIG. 10 is from an untreated cell. Neither forskolin (10 μM norgenistein (30 μM activated current. The top tracing in FIG. 10 is from acell that had been incubated in 5 mM 4-phenylbutyrate (4-PB) for twodays (Rubenstein et al., J. Clin. Invest. 100:2457-2465, 1997). After4-PB treatment, chloride current was stimulated by forskolin (by onaverage 30.3±19.4 pS/pF, n=6), and further activated by genistein (to anaverage 105±84 pS/pF) in a CF cell with the Δ508-CFTR mutation. Theseresults further demonstrate the ability of a flavenoid compound tooptimize chloride currents elicited in CF cells by other means.

Within another experiment, HeLa cells infected with theG551D-CFTR-containing adenovirus were investigated in the patch clampmode. Stimulation of the cell with forskolin (10 μM) stimulated only avery small current (FIGS. 11A and 11B). On average, forskolin-stimulatedconductance was 9.5±5 pS/pF (n=4). Additional stimulation with genistein(30 μM) stimulated significant chloride currents, which were time- andvoltage-independent (FIG. 11B) and well fitted with the Goldman equation(line in FIG. 11B; Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol.Physiol.):L902-910, 1998), which are characteristics of CFTR-mediatedcurrents. Average forskolin+genistein-activated conductance was 120±30pS/pF (n=4). Current variance to mean current plot (FIG. 11C) were usedto calculate the average open probability (P_(o) shown on top of axis)of the population of channels carrying the total current (as describedin Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol.Physiol.):L902-910, 1998). During forskolin stimulation, maximal P_(o)reached was 0.04 (open circles) and after additional stimulation withgenistein P_(o) reached a maximum of 0.42 in this recording. On average,after forskolin stimulation, P_(o)=0.05±0.02 and afterforskolin+genistein stimulation P_(o)=0.54±0.12. For comparison, wildtype CFTR expressed in HeLa cells and recorded under the same conditionsresulted in P_(o)=0.36±0.05 (n=3) after forskolin stimulation andP_(o)=0.63±0.16 after forskolin+genistein treatment.

Example 5 Effect of Representative Flavones on Nasal PotentialDifference in CF Patients

This Example illustrates the in vivo use of quercetin and genistein toactivate the nasal potential difference in CF patients bearing the G551Dmutation.

Measurements were performed on patients as described by Alton et al.,Eur. Respir. J. 3:922-926, 1990; Illek and Fischer, Am. J. Physiol.(Lung Cell. Mol. Physiol.):L902-910, 1998; and Knowles et al., Hum. GeneTherapy 6:445-455, 1995). The results are presented in FIGS. 12A and12B. FIG. 12A shows a recording from a patient with the genotypeG551D/ΔF508. Initial treatment with amiloride and chloride free solutionhad the purpose to isolate and amplify the chloride selectivepotentials. Addition of the beta-adrenergic agonist isoproterenol showedno effect, which is typical for CF patients (Knowles et al., Hum. GeneTherapy 6:445-455, 1995). However, addition of genistein hyperpolarizednasal PD. Average responses of nasal PD to genistein and quercetin offour CF patients with the G551D mutation are shown in FIG. 12B (openbars). The filled bars show for comparison the respective responses inhealthy subjects. The genotypes of the 4 CF patients were: twoG551D/ΔF508, one G551D/G551D and one G551D/unknown. Responses are mostlikely due to the G551D mutation because the homozygous G551D patientresponded not different compared to the heterozygous G551D patients.Genistein and quercetin responses of nasal PD in CF patients weresignificant (p<0.05).

These results demonstrate that CFTR mutations are sensitive to flavenoidtreatment, and provide additional evidence for therapeutic benefit ofsuch compounds for the treatment of cystic fibrosis.

Example 6 Effect of Additional Representative Polyphenolic Compounds onEpithelial Cell Chloride Currents

This Example illustrates the effect of further flavenoids andisoflavenoids on chloride currents in airway epithelial cells.

Airway epithelial cells were prestimulated with 10 μM forskolin. Thepercent increase in chloride current was then determined followingtreatment with a series of polyphenolic compounds. FIG. 13A summarizesthe stimulatory effect of these compounds. On average, chloride currentswere further stimulated by reservatrol (100 μM) to 135%, by flavanone(100 μM) to 140%, by flavone (200 μM) to 128%, by apigenin (20 μM) to241%, by apigenin 7-O-neohesperidoside (30 μM) to 155%, by kaempferol(20 μM) to 182%, by fisetin (100 μM) to 108%, by quercetin (30 μM) to169%, by rutin (30 μM) to 149%, by genistein (30 μM) to 229%, bydaidzein (50 μM) to 162%, by biochanin A (100 μM) to 139% and byprunetin (100 μM) to 161%.

The stimulatory effect of 7,4′ Dihydroxyflavone is shown in FIG. 13B.Addition of 7,4′-Dihydroxyflavone to the mucosal perfusiondose-dependently stimulated transepithelial C1 currents in unstimulatedCalu-3 monolayers. This experiment was performed using unstimulatedtissue.

The stimulatory effect of trimethoxy-apigenin is shown in FIG. 13C.Addition of trimethoxy-apigenin to the mucosal perfusiondose-dependently stimulated transepithelial C1 currents in unstimulatedCalu-3 monolayers. Kinetic analysis is depicted on the right panel andestimated half maximal stimulatory dose was 11.7 μM.

These results indicate that a variety of polyphenolic compoundsstimulate chloride currents in epithelial cells.

Example 7 Effect of Reservatrol on Chloride Currents

This Example illustrates the stimulatory effect of reservatrol ontransepithelial chloride currents.

Unstimulated Calu-3 monolayers were treated with increasingconcentrations of reservatrol. FIG. 14 shows the recording generatedfollowing the addition of reservatrol to the mucosal perfusiondose-dependently stimulated transepithelial chloride currents inunstimulated Calu-3 monolayers. For comparison, currents were furtherstimulated by serosal addition of forskolin. The stimulated chloridecurrent was completely blocked by the C1 channel blocker DPC. Theseresults indicate that reservatrol stimulates transepithelial chloridetransport.

Example 8 Effect of Ascorbic Acid and Dehydroascorbic Acid on ChlorideCurrents

This Example illustrates the stimulatory effect of ascorbic acid anddehydroascorbic acid on transepithelial chloride current.

Unstimulated Calu-3 monolayers were stimulated with L-ascorbic acid, asshown in FIG. 15. Addition of L-ascorbic acid to the mucosal or serosalperfusion very effectively stimulated transepithelial chloride secretionin unstimulated Calu-3 monolayers. For comparison, chloride currentswere further stimulated by serosal addition of forskolin. In thecontinued presence of L-ascorbic acid and forskolin, it is remarkablethat addition of genistein further stimulated chloride currents. Theseresults indicate that genistein serves as a potent drug that is able tohyperstimulate chloride secretion and thereby maximize chloridetransport across epithelia. The stimulated chloride current wascompletely blocked by the chloride channel blocker DPC.

The stimulatory effect of L-ascorbic acid is also shown in FIG. 16.Addition of 100 μM L-ascorbic acid to the mucosal or serosal perfusionvery effectively stimulated transepithelial chloride currents inunstimulated Calu-3 monolayers. For comparison, ascorbic acid-stimulatedchloride currents were stimulated by the cAMP elevating agonistforskolin (20 μM, serosal). Under these stimulated conditions kaempferolfurther hyperstimulated chloride currents. The stimulated chloridecurrent was completely blocked by the chloride channel blocker DPC (5mM).

The stimulatory effect of dehydroascorbic acid is shown in FIG. 18.Addition of dehydroascorbic acid at 10, 100 or 300 μM to the mucosal andserosal perfusion effectively stimulated transepithelial chloridecurrents in unstimulated Calu-3 monolayers. Stimulated C1 currentsreturned to baseline after 5-15 min.

Example 9 Effect of Ascorbic Acid on Chloride Currents In Vivo

This Example illustrates the stimulatory effect of ascorbic acid onhuman nasal potential difference.

Nasal potential difference measurement was performed on a humanvolunteer according to a protocol by Knowles et al., Hum. Gene Therapy6:445-455, 1995. Addition of L-ascorbic acid (100 μM) to the luminalperfusate in the nose (in the presence of amiloride (blocks Na currents)and in chloride-free solution) hyperpolarized nasal potential difference(PD) by 6.3 mV (FIG. 17). Addition of the β-adrenergic agonistisoproterenol further hyperpolarized nasal PD. Stimulation was reversedby washing out drugs with NaCl Ringer solution. These resultsdemonstrate the ability of ascorbic acid to stimulate chloride transportin epithelia in humans.

Example 10 Effect of Genistein on Chloride Currents in Mammary Epithelia

This Example illustrates the stimulatory effect of genistein in mammaryepithelial cells.

The stimulation of transepithelial short-circuit current (Isc) across31EG4 mammary epithelial monolayers by addition of 20 μM genistein isshown in FIG. 19. Na currents were blocked by mucosal addition ofamiloride (10 mM). Chloride currents were further stimulated byforskolin (20 μM, serosal). Currents were recorded in symmetrical NaClRingers solution at 0 mV and pulses were obtained at 2 mV.

The following Methods were used for the experiments described inExamples 11-16.

Human airway cell culture. The human submucosal serous gland-like cellline Calu-3, the CF nasal epithelial cell line homozygous for ΔF508 CFTR(CF15) and human tracheal primary cultures (hTE) were cultured asdescribed (Jefferson, D. M., Valentich, J. D., Marini, F. C., Grubman,S. A., Iannuzzi, M. C., Dorkin, H. L., Li, M., Klinger, K. W. & Welsh,M. J. (1990) Am. J. Physiol. 259, L496-L505. Sachs, L. A., Finkbeiner,W. E. & Widdicombe, J. H. (2003) In Vitro Cell Dev. Biol. Anim. 39,56-62.). Wildtype CFTR-corrected CF15 cells were generated viaadenovirus-mediated gene transfer. Growth media were nominally free ofascorbic acid (<10 μm, University Pathology Inc., Salt Lake City, Utah).For transepithelial measurements cells were grown on permeable filterinserts and used after 3 to 10 days.

Patch clamp analysis. Single channel patch clamp studies were performedon Calu-3 cells at 37° C. The outside-out recording mode was establishedvia the whole cell mode as described (Fischer, H. (2001) in Methods inMolecular Medicine, eds. Skatch, W. R. & Walker, J. M. (Humana PressInc, Totowa), pp. 49-66.). The bath solution contained (in mM) 145 NaCl,1.7 CaCl₂, 1 MgCl₂, 10 Hepes, 10 glucose, pH=7.4. The pipette solutioncontained (in mM): 15 N-methyl-D-glucamine (NMG) Cl, 10 EGTA, 1 MgCl₂,10 Hepes, 10 glucose, 120 NMG-gluconate, 5 MgATP, 0.1 LiGTP, pH=7.4.Open probabilities (P_(o)) for multi-channel recordings were calculatedfor consecutive 20-sec records with P_(o)═(I−I_(base))/(N·i), where I isthe average current of the respective record, I_(base) is theclosed-level current, N is the maximal number of channel levels observedin the total recording, and i is the single channel current.

Cyclic AMP measurements. Confluent Calu-3 cells were exposed toL-ascorbic acid or forskolin for 15 min and lysed with 0.1 M HCl.Cellular cAMP levels were measured in non-acetylated samples using acompetitive immunoassay for cAMP (R&D systems, Minneapolis, Minn.).

Short-circuit current measurement. Calu-3 or CF15 cells were grown asmonolayers, mounted in Ussing chambers and short-circuit current(I_(SC)) was recorded as described (Illek, B. & Fischer, H. (1998) Am.J. Physiol. 275, L902-L910). At 20-50 second intervals, transepithelialvoltage was clamped from zero to 2 mV and the transepithelial resistance(R_(te)) was calculated. A serosa-to-mucosa-directed Cl gradient wasapplied. Serosal Ussing chamber solution contained (in mM): 120 NaCl, 20NaHCO₃, 5 KHCO₃, 1.2 NaH₂PO₄, 5.6 glucose, 2.5 CaCl₂, 1.2 MgCl₂. Inmucosal Ussing chamber solutions, all Cl salts were exchanged forgluconate salts.

Nasal potential difference (NPD) measurements. Measurements of NPD wereperformed in healthy volunteers as described (Illek, B. & Fischer, H.,Supra). The study protocol was approved by the Internal Review Board atChildren's Hospital Oakland. Solutions and test drugs were perfused intoone nostril at ˜5 ml/min at room temperature (23-25° C.). NaCl solutioncontained (in mM): 145 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 Hepes, pH=7.4.In Cl free solutions all Cl salts were replaced by the respectivegluconate salts. All solutions were sterile filtered before use. NPD wassensed with an Ag/AgCl/agar electrode placed in the perfusing tube withrespect to an electrode placed on a slightly scratched skin part on theforearm (Alton, E. W. F. W., Currie, A. D., Logan-Sinclair, R., Warner,J. O., Hodson, M. E. & Geddes, D. M. (1990) Eur. Resp. J. 3, 922-926).

CF mice and rectal potential difference (RPD) measurements.Gene-targeted mice homozygous for the ΔF508 mutation (C57Bl6/J) receivedGolytely® (Braintree Laboratories, Inc.), a diarrhetic supplement, inthe drinking water to increase their lifespan (Clarke, L. L., Gawenis,L. R., Franklin, C. L. & Harline, M. C. (1996) Lab. Anim. Sci. 46,612-8). To correct the maturation defect of ΔF508 CFTR we used thechemical chaperone trimethylamine oxide (TMAO) (Brown, C. R.,Hong-Brown, L. Q., Biwersi, J., Verkman, A. S. & Welch, W. J. (1996)Cell Stress Chaperones 1, 117-25). CF mice were injected with 4 mg/gTMAO from a 4 M stock solution every 8 hours for 24 hours, which is aregimen to partially correct the ΔF508 defect in CF mice (Fischer, H.,Barbry, P., Illek, B., Sartori, C., Fukuda, N. & Matthay, M. A. (2001)Am. J. Physiol. 281, L52-L57). Controls were water-injected. The RPDassay was performed on mice that were anesthetized with 0.1 mg/g bodyweight ketamine and 0.01 mg/g body weight azepromazine. RPD was sensedwith a 1 M NaCl agar bridge inserted ˜1 cm into the rectum vs. asubcutaneous needle filled with 1 M NaCl. Measurements were performed inCl free solution containing 100 μm amiloride and 5 mM Ba(OH)₂ to isolatethe Cl-selective RPD.

RT-PCR analysis and cloning of SVCT2. Total RNA was isolated from airwaycultures grown on permeable filter inserts using the RNeasy Mini Kit(Qiagen, Oslo, Norway). All samples were treated with DNase (2 U DNase,Promega) and RNase inhibitor (100 U Superase-In, Ambion Inc.). RT-PCRwas performed using Superscript II RNase Reverse Transcriptase(Invitrogen) and 2.5 μm random hexamer primers (Applied Biosystems).First strand cDNA was used as a template in polymerase chain reactions(RedTaq DNA Polymerase, Sigma). The primer sequences for SVCT1 (103 bp)were forward: 5′-TTC TGG TTG TGC TGC TGA CC-3′ (SEQ ID NO:7); reverse:5′-TGT ATC AGA CCA CGC TCC TCT-3′ (SEQ ID NO:8). For SVCT2 (97 bp) thesequences were: forward: 5′-GCT GTT GCA CAC AGA ACA CA-3′ (SEQ ID NO:9);reverse: 5′-GAG GAG GCC GAT GAO TAO TTC-3′ (SEQ ID NO:10). Standard PCRwas performed using 30 cycles and annealing at 60° C. The cDNA codingfor the open reading frame of SVCT2 was PCR-amplified from hTE and wascloned in-frame into the XhoI and EcoRI restriction sites of theenhanced green fluorescent expression vector pEGFP/N1 (Clontech). Thisconstruct was transfected into CF15 cells.

Confocal Microscopy. The expression of SVCT2-EGFP fusion proteins wasassayed in filter-grown monolayers by confocal microscopy. ConfluentCF15 monolayers were fixed with 2% paraformaldehyde and immunostainedfor the tight junction protein ZO-1 as a marker of the apical regionusing an anti-ZO-1 antibody (BD Bioscience) and Alexa Fluor546-conjugated secondary antibody (Molecular Probes). Monolayers wereembedded in Crystal Mount (Biomedia) and observed with a 63×/1.4 NAoil-immersion objective. A 3-dimensional image was produced from a stackof Z sections at 1.1 μm intervals. Image stacks were deconvolved andvisualized using Huygens Professional Software by Scientific VolumeImaging (www.svi.nl).

Example 11 Vitamin C Activates CFTR Chloride Channels

The regulatory role of vitamin C on Cl ion channel activity was studiedin Calu-3 airway cells which express large amounts of native CFTR astheir major Cl conductance (Haws, C., Finkbeiner, W. E., Widdicombe, J.H. & Wine, J. J. (1994) Am. J. Physiol. 266, L502-L512). Using theoutside-out patch clamp mode it was found that L-ascorbic acid inducedopenings of CFTR Cl channels when applied to the extracellular surfaceof the patched membrane. In the recording shown in FIG. 20A, twochannels were activated by 100 μM L-ascorbic acid, and the averagesingle channel open probability (P_(o)) increased from zero to 0.21±0.08(n=4, FIG. 20B). Subsequent addition of 10 μM forskolin (acAMP-stimulating agonist) in the continued presence of L-ascorbic acidfurther stimulated Cl channel activity and average P_(o) increased to0.54±0.12. Details of the single channel recording are illustrated inFIG. 20C. The additional cAMP-induced activation of theascorbate-stimulated Cl channels did not alter its single channelamplitude or apparent gating kinetics. FIG. 20D shows thecurrent-voltage relationship of the ascorbate-stimulated chlorideconductance and the resulting slope conductance averaged 8.9±0.2 pS(n=4). At negative potentials no channel openings were resolvedindicating Cl-over-gluconate selectivity. Cyclic AMP is the majorintracellular messenger for the activation of CFTR. We tested thepossibility that L-ascorbic acid increased intracellular cAMP levels[cAMP], by either turning on cAMP production or preventing cAMPdegradation. FIG. 20E compares whole cell cAMP levels of Calu-3 cellschallenged with either increasing concentrations of L-ascorbic acid orthe cAMP agonist forskolin. Concentrations of forskolin of 10 nM orgreater increased [cAMP], dose-dependently (FIG. 20E, filled circles).In contrast, concentrations of L-ascorbate from 100 μM to 10 mM did notresult in a detectable increase of resting [cAMP], (FIG. 20E, opencircles), and a dose of 300 μM L-ascorbate did not alter the elevatedcAMP level that was stimulated by 100 nM forskolin (FIG. 20E, greycircle). These data suggest that L-ascorbic acid stimulated CFTRactivity by a cAMP-independent mechanism.

Example 12 Vitamin C Regulates Chloride Transport Across Human AirwaysIn Vitro and In Vivo

Vitamin C activation of CFTR-mediated Cl secretion was studied in Calu-3cells grown as epithelial monolayers (FIG. 21A). Exposure of the apicalmembrane to maximal concentrations of L-ascorbic acid stimulatedtransepithelial Cl currents (I_(SC)) in a sustained fashion to 96±11μA/cm² (n=22). Subsequent addition of forskolin further increased I_(SC)to 141±33 μA/cm² (n=9, p=0.029). On average, Cl secretion was stimulatedby ascorbate to 68% of the currents elicited by forskolin. The Clchannel blocker diphenylcarboxylate (DPC, 4 mM) was added as a measurefor the transcellular Cl current decreasing I_(SC) to 30±4 μA/cm². WhenCl secretion was first stimulated with forskolin (to 106±23 μA/cm², n=4)ascorbate had no significant effect on I_(se) (at 100 μM:ΔI_(Cl)=−1.5±3.5 μA/cm², n=4, not different from zero, one-sample ttest). Half-maximal stimulatory constant averaged 36.5±2.9 μm asdetermined from transepithelial dose-response experiments.Concentrations of >300 μM caused maximal stimulation (FIG. 21B), i.e.,concentrations significantly above the physiological plasmaconcentration (which saturate at ˜90 μM (Levine, M., Conry-Cantilena,C., Wang, Y., Welch, R. W., Washko, P. W., Dhariwal, K. R., Park, J. B.,Lazarev, A., Graumlich, J. F., King, J. & Cantilena, L. R. (1996) Proc.Natl. Acad. Sci. USA 93, 3704-3709).

In vivo effects of ascorbic acid on Cl transport were evaluated usingthe nasal potential difference (NPD) assay for Cl channel function. TheCl-selective NPD was measured in amiloride-containing (100 μm) andchloride-free solutions. These experiments served as an importantcontrol to verify the significance of the transepithelial resultsobtained with ascorbic acid in Calu-3 cells. In close agreement with thetransepithelial measurements, NPD was progressively hyperpolarizedduring the exposure of the nasal mucosa to L-ascorbic acid (by −6.7±0.4mV, n=3) and the cAMP-stimulating agonist isoproterenol (by −5.5±0.5 mV,n=4) (FIG. 21C). A dose of 300 μm L-ascorbic acid hyperpolarized NPD to72% of the NPD in presence of the cAMP agonist isoproterenol (10 μM).This in vivo assay showed that vitamin C activated Cl transport acrossthe nasal mucosa in human subjects similar to its in vitro effects ontransepithelial Cl currents.

Example 13 Function and Expression of Sodium-Dependent Vitamin CTransporters in Human Airway Epithelia

Ascorbate-stimulated Cl secretion was significantly reduced in theabsence of extracellular Na on the mucosal side (FIG. 22A). Furthermore,the response to ascorbate in Na-containing medium was blunted in thepresence of phloretin, a known inhibitor of the sodium-dependent vitaminC transporters SVCT1 and SVCT2 (Tsukaguchi, H., Tokui, T., Mackenzie,B., Berger, U. V., Chen, X. Z., Wang, Y., Brubaker, R. F. & Hediger, M.A. (1999) Nature 399, 70-75). These data suggested the involvement SVCT1and/or SVCT2 during the activation of ascorbate-stimulated Cl secretion.Molecular expression of SVCT1 and SVCT2 transcripts in Calu-3 cells wasverified using RT-PCR and DNA sequencing analysis. For comparison, humanciliated tracheal epithelial cultures (hTE) and a nasal cystic fibrosisairway cell line (CF15) were included. Comparison of the intensities ofthe specific PCR products suggested that SVCT2 was equally expressedamong all tested cell types (FIG. 22B, middle panel), whereas SVCT1 wasless abundant in Calu-3 and CF15 compared to hTE (FIG. 22B, top panel).DNA sequencing of the open reading frame of SVCT2 from hTE revealed thatSVCT2 from trachea (GenBank accession No. AY380556; SEQ ID NO:11) wasidentical to the published sequence from kidney (GenBank accession No.AJ269478; SEQ ID NO:12) with the exception of one nucleotide exchange atposition 1807 T->C. The recombinantly expressed SVCT2-EGFP fusionprotein was targeted exclusively to the apical membrane pole of CF15epithelia.

Example 14 Vitamin C is Specific for CFTR

The CF15 cell line was used to determine whether ascorbate-stimulated Clcurrents were solely mediated by the CFTR Cl conductance. The CF15 cellline is characterized by the absence of functional CFTR in the apicalplasma membrane but the presence of other non-CFTR Cl conductances(Jefferson, D. M., Valentich, J. D., Marini, F. C., Grubman, S. A.,Iannuzzi, M. C., Dorkin, H. L., Li, M., Klinger, K. W. & Welsh, M. J.(1990) Am. J. Physiol. 259, L496-L505). A concentration of L-ascorbicacid that lay within the upper plateau of the dose response curve (500μm) was applied and ascorbate-stimulated Cl currents in CF15 vs.wildtype CFTR-corrected CF15 monolayers was compared (FIG. 23A).L-ascorbic acid did not significantly increase I_(SC) (ΔI_(SC)=1.3±0.5μA/cm², n=5), whereas calcium-elevating agonists effectively stimulatedthe calcium-activated Cl conductance in these cells (not shown). Thedefective response to L-ascorbic acid was reversed in wildtypeCFTR-corrected CF15 epithelia such that I_(SC) responded promptly toL-ascorbic acid (ΔI_(SC)=7.5±0.5 μA/cm², n=6). The ascorbate-stimulatedcurrent was further activated by the cAMP agonist forskolin and totalstimulated I_(SC) averaged 12.1±1.1 μA/cm² (FIG. 4B). Theascorbate-stimulated Cl current reached 60±15% of theforskolin-stimulated current in CFTR-corrected CF15 monolayers, whichwas close to the corresponding findings in Calu-3 monolayers (68%, seeFIG. 21A). The absence of ascorbate-stimulated Cl currents in CF15monolayers supports the notion that non-CFTR Cl conductances were notactivated by L-ascorbic acid. These experiments demonstrated a causalrelationship between CFTR expression and ascorbate stimulation inepithelia.

Example 15 Stimulation of Rectal Potential Difference (RPD) in RescueCompound-Treated CF Mice In Vivo

In this Example, it was determined whether L-ascorbic acid affected thefunctional activation of ΔF508 CFTR in a CF-affected organism in vivo.Using gene-targeted mice homozygous for the ΔF508 mutation it was shownpreviously that the osmolyte trimethylamine oxide (TMAO) effectivelysupported ΔF508 CFTR trafficking in vivo (Fischer, H., Barbry, P.,Illek, B., Sartori, C., Fukuda, N. & Matthay, M. A. (2001) Am. J.Physiol. 281, L52-L57). Accordingly, the effect of L-ascorbic acid wastested in homozygous ΔF508 CF mice treated with TMAO by using the RPDassay as a functional end point measure for mutant ΔF508-CFTR function.FIG. 24A illustrates that TMAO-treated CF mice manifested a detectableresponse to a maximal dose of L-ascorbic acid (1 mM) or its epimerD-isoascorbic acid (300 μM) and RPD hyperpolarized on average by−3.2±0.8 mV (n=4) and −3.6±1.8 mV (n=3), respectively (FIG. 24B). Incontrast, perfusion with L-ascorbic acid or D-isoascorbic acid did notsubstantially alter RPD in water-injected control CF mice (ΔRPD=−1.1±0.4mV, n=9). TMAO treatment increased the ascorbate- andisoascorbate-stimulated RPD approximately 3-fold when compared tountreated CF mice (FIG. 24B) indicating the activation of outward Cl ionmovement through functionally restored ΔF508-CFTR channels in the rectalmucosa of CF mice. The results show that both L-ascorbate andD-isoascorbate are pharmacological tools for the activation ofΔF508-mutated CFTR after its trafficking defect has been corrected.

Example 16 Stimulation of Chloride Ion Transport by L-Ascorbate andD-Isoascorbate in Cystic Fibrosis Nasal Epithelia In Vitro

In this example, chloride currents stimulated by L-ascorbate orD-ascorbate were measured in CF15 versus. wildtype CFTR-corrected CF15monolayers. A concentration of L-ascorbate (1 mM) or D-isoascorbate (300μm) that lay within the upper plateau of the dose-response curve wasapplied to cells and the resulting chloride currents stimulated in CF15versus wildtype CFTR-corrected CF15 monolayers were measured (FIG. 25A-H). All experiments were performed in the presence of the sodiumchannel blocker amiloride. In untreated cystic fibrosis epithelia,chloride currents were not significantly stimulated by L-ascorbate(ΔIsc=1.1±0.3 μA/cm², n=13) (FIG. 25A) nor D-isoascorbate (ΔIsc=0.7±1.0μA/cm², n=7) (FIG. 25E). The defective responses to L-ascorbate andD-Isoascorbate were reversed after gene therapy such that wildtypeCFTR-corrected CF15 epithelia I_(Cl) responded promptly to L-ascorbate(ΔIsc=7.5±0.5 μA/cm2, n=6) or D-isoascorbate (ΔIsc=6.6±1.7 μA/cm², n=2).These experiments confirm the results described in Examples 14 and 15and demonstrate a causal relationship between CFTR expression and thechloride secretory response to both L-ascorbate and D-isoascorbate.

The functional activation of ΔF508 CFTR was determined after correctionof the underlying trafficking defect of ΔF508 CFTR usingS-Nitrosoglutathione (Zaman, et al., Biochem Biophys Res Commun 284:65-70, 2001; Snyder, et al., American Journal of Respiratory andCritical Care Medicine 165: 922-6, 2002; Andersson, et al. Biochemicaland Biophysical Research Communication 297(3): 552-557, 2002.). CF15monolayers were treated with 1 mM S-Nitrosoglutathione (SNOG) for 5-24hours to support trafficking of ΔF508 CFTR to the plasma membrane.SNOG-treated CF15 monolayers manifested a detectable response toL-ascorbate and D-isoascorbate and transepithelial chloride currentsincreased on average by 2.1±0.1, n=10 (L-ascorbate, FIG. 25C) and3.1±0.5, n=8 (D-Isoascorbate, FIG. 25G). SNOG treatment recovered theascorbate and isoascorbate-stimulated Cl secretion between 28-47% whencompared to wildtype CFTR corrected CF15 cells. The summary in FIGS. 25Dand 25G compares the magnitudes of the L-ascorbate and D-isoascorbatestimulated Cl currents in cystic fibrosis epithelial. These results showthat both L-ascorbate and D-isoascorbate are pharmacological tools forthe activation of ΔF508 mutated CFTRS after its trafficking defect hasbeen corrected.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All of references, patents,patent applications, etc. cited above, are incorporated herein in theirentirety. Further, all numerical ranges recited herein explicitlyinclude all integer values within the range.

What is claimed is:
 1. A composition comprising: (a) one or moreflavones or isoflavones capable of stimulating chloride secretion; (b)one or more of: (i) a compound that increases expression of a CFTRprotein in an epithelial cell; and (ii) a chemical chaperone thatincreases trafficking of a CFTR protein to a plasma membrane in anepithelial cell; (c) one or more of a compound selected from the groupconsisting of ascorbic acid, ascorbate salts, dehydroascorbic acid andresveratrol; and (d) a physiologically acceptable carrier.
 2. Thecomposition of claim 1 wherein the CFTR protein has a mutation atposition
 551. 3. The composition of claim 1 wherein the CFTR protein hasa ΔF508 mutation.